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Technical Briefs |
1 University Women’s Hospital/Department of Research, University of Basel, CH 4031 Basel, Switzerland
2 Division of Paediatric Nephrology, University of the Witwatersrand and Johannesburg Hospital, Johannesburg, South Africa
aaddress correspondence to this author at: Laboratory for Prenatal Medicine, University Women’s Hospital/Department of Research, Schanzenstrasse 46, CH 4031 Basel, Switzerland; fax 41-61-325-9399, e-mail shahn{at}unbs.ch
Recently, a novel form of chimerism, termed urinary DNA chimerism, has been described in kidney transplant recipients in that cell-free DNA from the donor kidney was detected in the recipient’s urine (1). Quantitative analysis of this urinary donor-derived DNA has indicated that it may serve as a new marker to monitor kidney transplant engraftment because increased concentrations were present under conditions of graft rejection, which decreased to basal values after immunosuppressive treatment (2). A caveat of these studies was that they relied on sex-disparate donor–recipient conditions: because the PCR assays used were specific for the Y chromosome, cell-free DNA from the donor kidney could be detected only in the urine of female recipients who had received male kidneys (1)(2).
We examined whether other kidney donor-derived DNA sequences could be detected in the urine of transplant recipients, using PCR assays specific for highly polymorphic short tandem repeat (STR) loci, also termed microsatellite markers. Previous examinations using such polymorphic genetic loci have shown that they can be used for differentiating female fetal cells from maternal ones (3)(4) or for the gender-independent detection of cell-free fetal DNA in maternal plasma (5)(6). For this purpose, we tested for the presence of donor-specific STR loci in the urine of cases in which the donor and recipient were either of the same sex or the donor was female and the recipient was male.
For our study, which was approved by our respective ethics review boards, five cases involving living-donor (four related and one unrelated) transplants were enrolled.
Blood samples from both the recipient and donor were obtained before the transplantation, and spontaneous urine samples were obtained from the previously anuric recipients post transplantation. Because there is some tentative evidence that DNA in urine can be stabilized by the presence of the chelating agent EDTA (7), the urine samples were collected and shipped in standard Monovette tubes (Sarstedt) used for the collection of blood samples (containing 1.6 mg of potassium EDTA/mL of total volume).
Whole-blood DNA and cell-free urinary DNA were extracted with use of the High Pure PCR Template reagent set (Roche), according to the manufacturer’s instructions.
The donor–recipient pairs were first genotyped using 100 ng of total genomic DNA to monitor microsatellite markers on chromosome 21 in a fluorescent PCR assay established previously in our laboratory (3):
The D21S11 and D21S1435 forward primers were 5'-labeled with the fluorescent dyes carboxyfluorescein (FAM) and 2,7-dimethyloxy-4,5-dichloro-6-carboxyfluorescein (HEX), whereas the D21S1432 and D21S1440 reverse primers were 5'-labeled with tetrachlorofluorescein (TET) and HEX, respectively. All primers were obtained form Microsynth Incorporated. This step allowed us to determine which of the STR loci on chromosome 21 were informative, in that a particular STR allele present in the donor genome was absent from that of the recipient. We should then be able to determine whether we could detect this donor-specific informative STR allele in the urine of the transplant recipient.
Because the concentration of cell-free DNA in urine was previously found to be very low (2), this material was examined by use of a seminested PCR assay we have used previously for the analysis of single cells (3). In this assay the following external seminested primers were used:
In brief, PCR amplification was performed in a total volume of 30 µL containing 100 ng of template DNA, 200 nM deoxynucleotide triphosphates, 10 pM each of the primers, 3.5 mM MgCl2, and 1.5 U of AmpliTaq Gold (Applied Biosystems Inc.). After denaturation at 95 °C for 10 min, PCR was performed for 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, with a final extension step at 72 °C for 7 min. For the urine samples, 1 µL of the PCR amplicon was used as template for a subsequent seminested PCR amplification. This nested PCR was performed as above except that the annealing temperature was increased to 58 °C. After amplification, the PCR products were analyzed by capillary electrophoresis on a ABI 310 gene analyzer (Applied Biosystems) equipped with GeneScan software (Applied Biosystems). Fluorescently labeled GeneScan 500 molecular weight markers were included in each run.
Our analysis of these STR loci showed that informative allelic differences could be obtained in each of the cases studied (Table 1
). These were then used to study the corresponding urine samples. Subsequent analysis showed that donor-specific STR alleles could be detected in each case examined (Table 1
and Fig. 1
). In general, the recipient urine samples contained both recipient- and donor-derived STR sequences (e.g., cases 1, 2, and 5) in that informative donor and recipient alleles could be detected in these samples. In one recipient (case 3), donor-derived sequences appeared to dominate in that the informative recipient allele was lacking. In case 4, the recipient was homozygous for both of the STR markers tested. In the urine of this patient, however, the unique donor-derived STR allele as well as the allele common to both donor and recipient were detectable for both the STR markers examined.
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Because our investigation used a nested PCR assay in which a post-linear amplification phase amplicon was reamplified, no statement concerning the relative quantities of the donor and recipient cell-free DNA species is possible. For this reason, we examined two genetic polymorphisms in the glutathione S-transferase M1 (GSTM1) and angiotensin-converting enzyme (ACE) genes, recently described for the quantitative analysis of fetomaternal cell traffic and transfer of cell-free DNA (8). Unfortunately, in our study, we were able to obtain an informative constellation only in a solitary instance for only one of these loci, i.e., the GSTM1 gene, in which instance the gene was absent from the recipient. Of interest is that this case involved the transplantation of a kidney from an unrelated donor. Our analysis of this sample indicated that the recipient’s urine contained >77 000 copies of cell-free donor-derived DNA/mL immediately post transplantation, which decreased to slightly more than 100 copies/mL of urine by day 7. The concentration of total cell-free DNA was initially determined to be >92 000 copies/mL of recipient urine, which decreased to 560 copies/mL of urine by day 7, based on a real-time PCR assay for the GAPDH gene (9). This analysis indicated that almost all of the cell-free DNA in the recipient urine was donor-derived, a feature that is in good accord with previous reports (1)(2).
The limited usefulness of the polymorphic ACE and GSTM1 loci in our study could be a reflection of the rather small study size (only five cases). Nevertheless, it does indicate that assays for other markers will need be developed in the future to guarantee effective analysis of all donor–recipient constellations. Because we were readily able to detect informative donor-derived STR alleles in all of the samples tested, our results do suggest that it should be possible to detect other polymorphic markers more amenable to quantification by real-time PCR.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. Li, D. Hahn, W. Holzgreve, and S. Hahn Ready Detection of Donor-Specific Single-Nucleotide Polymorphisms in the Urine of Renal Transplant Recipients by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Clin. Chem., October 1, 2005; 51(10): 1903 - 1904. [Full Text] [PDF]
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Y. Li, B. Zimmermann, C. Rusterholz, A. Kang, W. Holzgreve, and S. Hahn Size Separation of Circulatory DNA in Maternal Plasma Permits Ready Detection of Fetal DNA Polymorphisms Clin. Chem., June 1, 2004; 50(6): 1002 - 1011. [Abstract] [Full Text] [PDF]
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 Y. Li, X. Y. Zhong, A. Kang, C. Troeger, W. Holzgreve, and S. Hahn Inability to Detect Cell Free Fetal DNA in the Urine of Normal Pregnant Women nor in Those Affected by Preeclampsia Associated HELLP Syndrome Reproductive Sciences, December 1, 2003; 10(8): 503 - 508. [Abstract] [PDF]
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